Method for homing hematopoietic stem cells to bone marrow stromal cells

ABSTRACT

This invention pertains to a method for homing hematopoietic stem cells to bone marrow stromal cells in a host. The method comprises, administering to the host genetically-engineered hematopoietic stem cells capable of expressing a first member of a ligand-receptor binding pair. The stem cells are administered to the host under conditions whereby binding of the first member of the ligand-receptor binding pair to the second member of the ligand-receptor binding pair, present on stromal cells, occurs thereby homing the stem cells to the stromal cells. This method is useful for transplanting bone marrow in a host or in treating a host afflicted with a disease associated with a disorder of the bone marrow.

GOVERNMENT SUPPORT

[0001] Work described herein was supported by grants from the NationalInstitutes of Health and the National Cancer Institute.

BACKGROUND OF THE INVENTION

[0002] Long-term bone marrow cultures (LTBMC) contain two major cellpopulations referred to as compartments. The hematopoietic stem cellcompartment, contains cells at various stages of self renewal capacityand differentiation and the adherent cell or stromal cell compartmenthas been shown to provide the environment necessary for the productionand differentiation of hematopoietic stem cells and their progenitors.Both compartments interact to facilitate hematopoiesis in vitro(Naparstek, et al; Exp. Hematol. 13: 701-708 (1985).

[0003] Bone marrow transplants are widely used for treating congenitaldisorders of the bone marrow or hematopoietic stem cell, e.g. aplasticanemia, acute leukemias, recurrent lymphomas, or solid tumors. Prior toreceiving a bone marrow transplant, the recipient is prepared byablating or removing endogenous recipient hematopoietic stem cells. Thispreparation is usually carried out by total body irradiation or deliveryof a high dose of an alkylating agent or other chemotherapeuticcytotoxic agents (Greenberger, J. S., Br. J. Hematol, 62: 606-605, 1986;Anklesaria, P., et al, PNAS, USA, 84: 7681-7685, 1987; Thomas, E. D.,Cancer, 49: 1963, 1982; Thomas, E. D., N. Eng. J. Med., 292: 832-843,895-902, 1975). Following preparation of the recipient, donor bonemarrow cells are injected intravenously and have been demonstrated tohome to multiple sites within the recipient where they proliferate andreconstitute all elements of the hematopoietic stem cell compartmentincluding neutrophilic granulocytes, megakaryocytes (platelets), redblood cell progenitors leading to mature erythrocytes, T-lymphocytes,B-lymphocytes, monocyte/macrophages, basophils and mast cells (Thomas,E. D., cited supra). Some data from several clinical transplanationcenters suggest that donor origin stromal cells of the hematopoieticmicroenvironment are also detected in small numbers in recipients aftermarrow transplant (Thomas, E. D., cited supra).

[0004] Two general categories of marrow transplanation have beendescribed. In an allogeneic transplant, HLA tissue typing is carried outon various marrow donors and a matched marrow specimen as close aspossible to that of the recipient, is used as the donor cell population.Allogeneic marrow transplant is the most common form of transplant inpatients with malignancy of the marrow compartment where removal of themalignant cells from the marrow is a very difficult process (Thomas, E.D., cited supra).

[0005] The other category of bone marrow transplant is autologous marrowtransplant. An autologous marrow transplant involves removal of thepatient's own bone marrow and washing or preparing it by techniques thatremove unwanted populations of cells (including tumor cells). The washedcells are then reinfused into the patient after the preparative regimenis completed. Under these conditions, the problems of graft versus hostdisease, or rejection of non-matched marrow can be reduced oreliminated. Thus, there is a decreased risk of infection or graftfailure.

[0006] Autologous marrow transplant is gaining popularity and frequencythroughout the United States and Europe for the treatment of solidtumors or recurrent lymphomas. For these treatments, higher doses ofchemotherapy and radiation therapy can be delivered. For this technique,there must be a source of untreated marrow available to give back to thepatient. Autologous marrow transplant is generally the safer form ofbone marrow transplantation because it overcomes many of the immune,histocompatibility, and rejection problems. Further, an autologousmarrow transplant requires less of a support facility for a new hospitalor treatment center setting up such a program (Thomas, E. D., citedsupra).

[0007] Graft failure is a common complication of marrow transplantationwith both autologous and allogeneic protocols (Thomas, E. D., citedsupra). The mechanism of graft failure has been studied for many years.Clinical research data presented at national and international meetingsover the last ten years has pointed toward a defect in bone marrowstroma as the cause of graft failure. Such a defect in marrow stroma maybe attributable to the preparative regimen of total body irradiationand/or chemotherapy that is used to prepare the patient for thetransplant (autologous or allogeneic). In some diseases, such as chronicmyelogenous leukemia, a defect in marrow stroma can be an inherent partof the disease process.

SUMMARY OF THE INVENTION

[0008] This invention pertains to a method for homing hematopoietic stemcells to bone marrow stromal cells in a host comprising, administeringto the host genetically-engineered hematopoietic stem cells capable ofexpressing a first member of a ligand-receptor binding pair. The stemcells are administered to the host under conditions whereby binding ofthe first member of the ligand-receptor binding pair to the secondmember of the ligand-receptor binding pair, present on stromal cells,occurs thereby homing the stem cells to the stromal cells.

[0009] Another embodiment of the method for homing hematopoietic stemcells to bone marrow stromal cells in a host can comprise a first stepof administering to the host stromal cells capable of expressing a firstmember of a ligand-receptor binding pair followed by a second step ofadministering hematopoietic stem cells capable of expressing a secondmember of a ligand-receptor binding pair. In this embodiment, either thestem cells or stromal cells are genetically-engineered to provide thecapability of expressing the appropriate ligand or receptor. The methodsof this invention can be used for transplanting bone marrow in a host orfor treating a host afflicted with a disease associated with a disorderof the bone marrow.

[0010] Transplantation of a donor bone marrow microenvironment,including both the stromal and stem cells, can reduce the risk of graftfailure in instances where a defect in a patient's stroma is causing thegraft failure. A problem encountered when attempting to transplant twocell lines which are dependent on each other is that there are multiplepossible sites in a host where the cells can, “home”. Thus, there is achance that the cell lines may not “home” together. The method of thisinvention provides a method for homing stem cells to the engraftedmicroenvironment thereby allowing proliferation of the stem cells atthese new sites to produce all the formed elements of the blood. Themethod of homing stem cells to stromal cells is advantageous in that itcan be used to reconstitute the marrow of patients who have damagedmarrow stroma and stem cells due to injury, congenital defects, orcytotoxic therapy.

DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 is a schematic showing the transfection of a stromal cellline with an expression vector containing the Mo-MuLV LTR, the entirecoding region (595 bp) for the mature 50 amino-acid transforming growthfactor α (TGFα), and the neomycin gene which confers G418 resistance tomammalian cells. The transfection of a hematopoietic cell line with anexpression vector containing cDNA for epidermal growth factor receptor(EGF-R), the Mo-MuLV LTR, and the E. coli GPT gene which confersresistance to mycophanolic acid is also depicted in the schematic.

[0012]FIG. 2 is a photograph of a Northern blot analysis of total mRNAfrom the GPTGFα (GPTα), GBL/6 and EuT cell lines, for detectabletranscripts for TGFα. EuT cells were used as the positive controls.Total mRNA (10 μgs/lane) was run on a 1% agarose gel in 1% formaldehydeand transferred to a nylon membrane. Filters were hybridized with a ³²Plabelled specific cDNA (542 bp insert), washed at high stringency andautoradiograms exposed for three to four days at −70° C. withintensifying screens. A 4.8 Kb specific message was detected in theGPTGFα cells compared to the GBL/6 cells. This data, in addition to thebiochemical evidence for release of TGFα in the conditioned medium ofGPTGFα cells, provides strong evidence for the expression of TGFα in theGBL/6 stromal cell line.

[0013]FIGS. 3A and 3B are graphs depicting the support of 32D-EGFR cellsby a TGFα producing GPTGFα stromal cell line. The 32D-EGF-R cells(2.5×10⁶) were cocultivated with confluent cultures of GBL/6 or GPTGFαcells. At weekly intervals, “cobblestone areas” were scored andnon-adherent cells were harvested and counted. The flasks were refed byadding an equal volume of fresh medium. The results are expressed as themean ±SD of three flasks per experiment. FIG. 3A shows the cumulativenumber of “cobblestone areas” per flask for over fifty days in culture.FIG. 3B shows the cumulative production of non-adherent cells/flask forover fifty days in culture. Factor-dependent 32D-EGF-R cells platedwithout interleukin-3 (IL-3) or epidermal growth factor (EGF) or stromalcells were not viable at four days in culture and did not adhere toplastic. There was a strong and clear effect that GPTGFα cells support32D-EGFR cells in vitro in the adherent population and growth afterdetachment.

[0014]FIG. 4 is a schematic depicting the transplantation protocol.Adult mice received 3.0 Gy (TBI) and in addition irradiation to bothhind limbs of 10.0-12.5 Gy, delivered by a 6 MeV Siemans linearaccelerator. During irradiation mice were restrained in a lucite holderdesigned to shield non-boosted areas. The dose rate at severalirradiated sites was measured by thermoluminescent dosimeters. The doserate for the TBI ranged from 0.25-1.0 Gy/minute and to the exposed limbfrom 1.0-1.15 Gy/min.

[0015]FIG. 5 is a scan of ¹¹¹In-32D or In-fresh bone marrow (FBM) cellsin vivo. Factor dependent-32D cells or fresh bone marrow cells werelabelled for twenty minutes with ¹¹¹In-Oxine (300 μCi/10 cells). Thelabelled cells were injected intravenously into lethally irradiated(1000 cGy) C57BL/6 mice. The mice were anaesthetized twenty-four hoursposttransplant by using an inhalation anesthetic, methoxyflurane.Subsequently, the mice were scanned using a gamma camera (Picker)connected to a PDP-11 digital computer for twenty minutes. The arrowsindicate localization of labelled cells (FBM) but not 32D cells to thehigh dose irradiated right hind limb as well as the liver and spleen(central dark area).

[0016]FIG. 6 is a photograph of 32D-EGFR cells forming foci of flattenedadherent cells.

[0017]FIG. 7 is a set of graphs depicting the inhibition of 32D-EGFRcell adhesion by soluble EGF receptor ligands.

[0018]FIG. 8 is a photograph depicting 5-bromo-2′ deoxyuridine (BUdR)labeling of replicating nuclei in adherent 32D-EGFR cells.

Detailed Description

[0019] This invention pertains to a method for homing hematopoietic stemcells to bone marrow stromal cells in a host. The method comprisesadministering to the host genetically engineered hematopoietic stemcells capable of expressing the first member of a ligand receptorbinding pair under conditions whereby binding of the first member of theligand receptor binding pair to a second member of the ligand receptorbinding pair, present on stromal cells, occurs thereby homing the stemcells to the stromal cells.

[0020] When it is desired to replace the stroma in a host, the methodcomprises a step of administering to the host stromal cells capable ofexpressing a first member of a ligand receptor binding pair followed byhematopoietic stem cells capable of expressing a second member of aligand receptor binding pair. The cells are administered underconditions whereby binding of the ligand to the receptor occurs therebyhoming the stem cells to the stromal cells.

[0021] In this embodiment, either the stem cells or the stromal cellsare genetically-engineered to provide the capability of expressing theappropriate ligand or receptor. When the methods are used to treat ahost afflicted with a bone marrow associated disease, a therapeuticallyeffective amount of stromal cells capable of expressing a first memberof a ligand receptor binding pair and a therapeutically effective amountof hematopoietic stem cells capable of expressing a second member of theligand receptor binding pair are administered to the host.

[0022] Hosts which can be used within this invention are animalssusceptible to bone marrow disorders or which may need a bone marrowtransplant (i.e., animals which have bone marrow). Examples of hostsinclude humans and domestic animals (e.g., dogs, cats, and horses).

[0023] The hematopoietic stem cells and bone marrow stromal cells arepreferably derived from the species of host being treated or can bederived from a species which does not invoke significant immuneresponses in the host. The stem cells or stromal cells can also bederived from the host if the cells are functioning properly. The stromaland/or stem cells can be removed from the host and cultured usingconventional techniques. Examples of human haematpoietic stem cell lineswhich can be used in this invention include nonadherent cells derivedfrom human long-term bone marrow cultures. See Greenberg, H. M., et al,Blood, 1981, Vol. 58, pp 724-732, the contents of which are herebyincorporated by reference. Examples of human bone marrow stromal cellsinclude KM101, KM102, KM103, KM104 and KM105. See Fitzgerald et al, Int.J. Radiation Oncology Biol. Phys. Vol 15, pp 1153-59 (1988), thecontents of which are hereby incorporated by reference.

[0024] The hematopoietic stem cells or stromal cells can begenetically-engineered using conventional techniques. The DNA encodingthe desired ligand or receptor can be inserted into a vector andintroduced unto the cells using techniques such as electroporationand/or retroviral infection. Other techniques which can be used tointroduce DNA into the cells are calcium phosphate precipitation (Grahamand van der Eb, Virology 52:456 (1973) and DEAE-dextran (Cullen et al.,Nature 307:241 (1984)).

[0025] The ligand-receptor binding pair are substances having anaffinity for each other. At least one member of the ligand-receptor pairis proteinaceous. Examples of ligand-receptor binding pairs includetransforming growth factor (TGF) and transforming growth factor receptor(TGFR) or EGF Receptor; (EGFR) epidermal growth factor (EGF) and EGFR;tumor necrosis factor-α (TNF-α) and tumor necrosis factor-receptor(TNFR); interferon and interferon receptor; platelet derived growthfactor (PDGF) and PDGF receptor; transferrin and transferrin receptor;avidin and biotin or antibiotin; antibody and antigen pairs; interleukinand interleukin receptor (including types 3, 4 and 5);granulocyte-macrophage colony stimulating factor (GMCSF) and GMCSFreceptor; macrophage colony stimulating factor (MCSF) and MCSF receptor;and granulocyte colony stimulating factor (G-CSF) and G-CSF receptor.Further, the ligand-binding pair can be a pair wherein the first memberis naturally-occurring and the second member is provided usinggenetic-engineering techniques. For example, the stromal cells can begenetically-engineered by inserting DNA encoding sugar receptors andthis will enhance the homing of the stem cells to the stromal cellsbased upon the naturally-occurring sugar molecules present in stem cells(Aizawa et al; Exp. Hematol. 16: 811-813 (1988).

[0026] The terms ligand and receptor are intended to encompass theentire ligand or receptor or portions thereof. Portions which can beused within this invention are those portions sufficient for binding tooccur between the ligand and the receptor.

[0027] The cells can be administered by subcutaneous or other injectionor intraveneously. In methods for treating a host afflicted with a bonemarrow associated disease, a therapeutically effective amount of stemcells or stromal cells is that amount sufficient to significantlyreduced or eliminate the symptoms or effects of a bone,marrow associateddisease. The therapeutically effective amount administered to a hostwill be determined on an individual basis and will be based, at least inpart, on consideration of the individual's size, the severity ofsymptoms to be treated, and the results sought. Thus, a therapeuticeffective amount can be determined by one of ordinary skill in the artof employing such practice in using no more than routineexperimentation.

[0028] This invention will be further illustrated by the followingexample.

EXAMPLE 1 Generation of a Bone Marrow Stromal Cell Line and PurifiedHematopoietic Stem Cells

[0029] Human bone marrow stromal cell lines can be established using thetechnique described by Fitzgerald et al (1988) cited supra and Harigayaet al, PNAS USA 83: 3477-3488 (1985). Human stem cells can be purifiedfrom long-term bone marrow cultures using the techniques described inGreenberg, H. M., et al, Blood, 1981, Vol. 58, pp 724-732, the contentsof which are hereby incorporated by reference.

Transfection and Isolation of Cell Lines

[0030] The vectors, pZipTGFα and pZipSV(x), were constructed aspreviously described (Finzi et al., PNAS USA, 84: 3733-37, (1987);William et al, Nature, 310: 476-78 (1984). Murine GBl/6 stromal cellswere transfected with pZipTGF-α using electroporation as described byPierce et al. (Science, 239: 628-31 (1988)). After twenty-four hours,the medium was replaced and the cells were selected for pZipTGFαtransfectants in 1 mg/ml G418 (Gieneticin GIBCO). Cells which wereresistant to G418 were expanded and assayed for TGF-α production. Thecells containing the DNA encoding TGF-α were labeled GP-TGF-α. A controlcell population was generated by defective retroviral vector infectionof the urine GBl/6 cell line using 24-hour culture supernatants from 4₂cells transfected with the PZip/neo DNA. This control cell populationwas labeled GBlneo®.

[0031] Transfected and infected cells were selected for resistance toG418 (1 mg/ml) and expanded. All cells were grown in Dulbecco's modifiedEagle's medium supplemented with 10% fetal bovine serum and 10 μMhydrocortisone sodium succinate. The generation of ZipTGF cells byinfection of NIH-3T3 mouse fibroblasts with pZip-TGFα has been described(Finzi et al, cited supra; DiMarco et al, Oncogene 4, 831-838 (1989)).ZipTGF clone EUT was used in the present examples.

[0032] Transfection and selection of IL-3-dependent murine 32D stemcells with a retroviral vector construct containing the human EGFreceptor cDNA has been described (Pierce et al, Science 239, 628-6311988). 32D-EGFR cells respond to EGF at concentrations ranging from 0.15nM to 5 nM. 32D-EGFR cells and parental 32D cells were maintained assuspension cultures in RPMI 1640 medium supplemented with 10% fetal calfserum and EGF (Pierce et al, cited supra) or IL-3 (Greenberger et al,Fed. Proc. 42, 106-115 1983; Ohta et al, Pathol. Immunopathol. Res. 8,1-20 1989) as previously described.

Generation of a Stromal Cell Line that Expresses pro TGF-α on the CellSurface

[0033] The murine bone marrow stromal cell line, GBl/6, supportsmyelopoiesis of enriched progenitors from long-term bone marrowcultures, but does not support adhesion or proliferation of theinterleukin-3 (IL-3)-dependent hematopoietic progenitor cell line, 32D(Anklesaria et al, Proc. Natl. Acad. Sci. USA 84, 7681-7685 1987);Greenberger et al, cited supra). GB1/6 cells were transfected with theretrovirus expression vector pZip-TGFα containing the entire codingregion for human proTGF-α transcribed under the control of a retroviralLTR (Finzi et al, cited supra (1987)) to obtain a GB1/6 derivative thatwould express pro- TGF-α. The same vector lacking the proTGF-α cDNAinsert, pZIP/neo, was introduced into GBl/6 to generate a control cellpopulation. The resulting cells expressing these vectors were designatedGP-TGF-α and GBlneo®, respectively, and were selected for resistance toG418 conferred by expression of the bacterial transposon Tn5 neomycinresistance gene (Cepko et al, Cell 37:1653-62 (1984)) present in thevectors. Both cell mass populations were expanded and analyzed for theirability to express proTGF-α. Northern blot analysis demonstrated thepresence of proTGF-α mRNA with the expected 4.8 kb size in GP-TGF-αcells, and no detectable expression of the endogenous TGF-α gene inGBlneo® cells (FIG. 2).

[0034] The Northern Blot Analysis was conducted as follows. PolyA+ RNAwas isolated from samples of at least 10⁸ cells by lysis in the presenceof proteinase K and sodium dodecyl sulfate as previously described(Badley et al, Biofeedback 6:114-116 (1988)). Samples of polyA+ RNA wereelectrophoresed in a 1% agarose gel, blotted onto-α nylon membrane andprobed with a human TGF-α cDNA radiolabeled by random-priming, aspreviously described (Finzi et al, cited supra (1987)).

[0035] The biosynthetic processing of proTGF-α in GP-TGF-α cells wasevaluated by pulse chase metabolic labeling experiments. Cells werelabeled with ³⁵S-cysteine for fifteen minutes followed by incubationwith unlabeled regular medium for various time lengths. Lysates obtainedfrom these cells were immunoprecipitated with antibodies raised againsta synthetic peptide that corresponds to a C-terminal cytoplasmicsequence of proTGF-α (Teixido et al, Nature 326, 883-885 1987).Polyacrylamide gel electrophoresis and fluorography of theseprecipitates displayed a proTGF-α biosynthesis pattern very similar tothat observed in other cell lines that express proTGF-α (Bringman et al,1987; Gentry et al, 1987, Wong et al, Cell 56, 495-506 1989). Thus,proTGF-α appeared as two products of 17 kd and 21 kd immediately afterthe labeling pulse. These two products disappeared after fifteen minutesof metabolic chase concomitantly with the appearance of a 18 kd labeledproduct (FIG. 2B). Based on previous characterization of similarproducts from other cell lines that express proTGF-α, these productswere identified as nascent proTGF-α (17kd), fully glycosylatedpost-Golgi proTGF-α (21kd), and proTGF-α cleaved at the N-terminaldomain that precedes the TGF-α sequence in the precursor (18 kd).

[0036] The level of expression of proTGF-α in GP-TGF-α cells was too lowto allow detection of this molecule on the cell surface by labeling with¹²⁵ and immunoprecipitation. To determine whether proTGF-α becameexposed on the surface of GP-TGF-α cells, susceptibility to cleavage byelastase was tested. Elastase cleaves at the N-terminus of the TGF-αsequence (Ignotz et al, Proc. Natl. Acad. Sci. USA 83, 6307-6311 1986).Thus, GP-TGF-α cell monolayers radioactively labeled under conditionsthat preferentially label the 21 kd proTGF-α species were exposed toelastase for one hour at 4° C. This treatment resulted in quantitativeconversion of the 21 kd labeled proTGF-α species to a 18 kd labeledproduct (FIG. 2C). This effect of elastase was dependent on the time ofincubation and was not observed in control cultures that receivedelastase immediately before the incubation was stopped and the sampleswere prepared for immunoprecipitation.

[0037] These results indicated that proTGF-α synthesized in GP-TGF-αcells became rapidly exposed on the cell surface and cleaved at theN-terminus. Further processing of the molecule was a very slow process.Consistent with these results, the concentration of free TGF-α in mediumconditioned for twenty four hours by GP-TGF-α was very low, below thedetection limit (20 pM) of our radioreceptor assay (Wong et al., Cell56:495-506 (1989)) and radioimmunoassay (Ignotz et al, 1986, citedsupra). By comparison, ZipTGF (clone EUT) cells that derive from NIH-3T3cells by transfection with pZip-TGFα and express a high level ofproTGF-α mRNA (Finzi et al, 1987, cited supra; and FIG. 1A) accumulated15 nM TGF-α in the medium in twenty-four hours.

Pulse Labeling and Immunoprecipitations

[0038] For pulse labeling studies, subconfluent GP-TGF-α and GBlneo®cell monolayers were pulse labeled for 20 minutes with 300 μCi/ml of^(±)S-cysteine (DuPont-New England Nuclear) in cysteine-free andserum-free Modified Eagle's Medium (MEM). The pulse label was chased byaddition of complete MEM. At the indicated times, cells were rinsed onceand scraped into immunoprecipitation buffer consisting of 130 mM NaCl,20 mM sodium phosphate, 1 mM EDTA, 1 mM PMSF and 200 KIU/ml ofaprotinin, pH 7. Cell pellets collected by centrifugation were thenlysed in the same buffer containing 1% Nonidet P-40. In preparation forimmunoprecipitation, cell lysates clarified by centrifugation at12,000×g for ten minutes were reduced and alkylated by incubation for 20minutes at 22° C. with 5 mM dithiothreitol and 0.25% sodium dodecylsulfate (SDS), followed by addition of 10 mM N-ethylmaleimide andincubation for 10 minutes at 22° C. Anti-pro TGF-α IgG fraction was thenadded at a 1/25 dilution relative to the original antiserum, and themixture was incubated overnight at 4° C. To assess specificity of theimmunoprecipitated products, C-terminal proTGF-α synthetic peptidesagainst which the antibodies had been raised (Teixido et al, 1987, citedsupra). were added at a final concentration of 5 mM to theimmunoprecipitation reactions. Immuno-complexes were harvested withprotein A-Sepharose and washed three times with phosphate bufferedsaline (PBS) containing 0.1% Triton and 0.025% SDS, and once with PBSalone. The washed beads were heated in electrophoresis sample buffer for5 min at 100° C., and were electrophoresed on 12% to 18% gradientpolyacrylamidedodecyl sulfate gels followed by fluorography usingEnlightning (DuPont-New England Nuclear).

[0039] For elastase treatment, GP-TGFα cells labeled with 300 μCi/ml of³⁵S-cysteine for twenty minutes and chased for five minutes withcomplete medium at 37° were chilled on ice. Cell monolayers were washedwith ice-cold MEM and incubated for 1 hour at 4° C. with MEM with orwithout 250 μg/ml of porcine pancreatic elastase (Worthington) and 250μg/ml of soybean trypsin inhibitor (Sigma) to prevent proteolysis bytrypsin that might contaminate the elastase preparation. Controlcultures received elastase immediately before stopping the incubation.Incubations were stopped by washing the cell monolayers three times withimmunoprecipitation buffer. Samples were then immunoprecipitated withani-pro TGF-α antibodies as described above.

EGF Radioreceptor Assay

[0040] Radioreceptor assays to measure soluble TGF-α in samples ofconditioned medium were performed using low density cultures of A431cells in 2 cm² wells and ¹²⁵I-EGF as the tracer radioligand. PurifiedEGF and TGF-α were used as standards. Experimental samples and standardswere subjected to the assay either directly or after 20-foldconcentration by dialysis against 0.1 M acetic acid, lyophilization andresuspension in assay medium. At the end of the assays, cells weresolubilized with a 1% Triton X-100 solution and counted for ¹²⁵radioactivity. Other assay conditions were as previously described (Wonget al., 1989, cited supra).

Homing of A Genetically-Engineered Hematopoietic Cell Line Expressingthe EGF Receptor to Stromal Cells Expressing proTGFα

[0041] The hematopoietic progenitor murine cell line 32D is dependent onIL-3 for proliferation and survival, and does not respond to otherhematopoietic growth factors including GM-CSF and CSF-1 (Greenberger etal, cited s_; Ohta et al, cited supra). This cell line lacks EGFreceptors but expresses all the components of the intracellular pathwayneeded to mediate a mitogenic response to this factor, as demonstratedwith the EGF receptor-transfected 32D cell clone 32D-EGFR (Pierce et al,Science 239: 628-631 1988). For these reasons, the 32D-EDFR cell linewas chosen to test whether the EGF receptor/proTGF-α pair could mediatecell-cell adhesion and lead to a mitogenic response.

[0042] 32D-EGFR cells were cocultivated with confluent monolayers ofGP-TGF-α stromal cells in the absence of any added IL-3 or EGF. Withinfour to six days, the 32D-EGFR cells began to form foci of flattenedadherent cells with approximately 10 cells/focus (FIG. 6). Themorphology of these foci was typical of the “cobblestone islands”generated by primary cultures of bone marrow haematpoietic progenitorsand stromal cells (Dexter et al, J. Cell. Physiol. 91, 335-344 1977;Williams et al, J. Cell. Physiol. 102, 287-295 1977; Greenberger, citedsupra; Anklesaria et al, cited supra). The 32D-EGFR cell islandsprogressively increased in size (>25 cells/island) and number betweendays 6 and 40 of cocultivation (FIG. 3A, a bars). Cell adhesion andisland formation were not detected when 32D-EGFR cells were cocultivatedwith GBlneo® cells, or when 32D cells were cocultivated with GP-TGF-αcells or GBlneo cells® (FIG. 3A, bars b, c and d).

[0043] In addition to attachment to the stromal layer, the adherent fociof 32D-EGFR cells were able to continuously release viable hematopoieticcells into the culture medium- for at least forty days in culture (FIG.3B, α bars). Cells released into culture medium had the phenotype ofnormal 32D-EGFR cells (Pierce et al, 1988, cited supra) as determined bytheir ability to respond to both EGF and IL-3, and to form colonies insemisolid medium (data not shown). In contrast, there were no viablecells (FIG. 3B, bars b and d) or less than 1% of the initial innoculum(FIG. 3B, c bars) produced when hematopoietic-stromal cells werecocultivated in combinations other than 32D-EGFR/GP-TGF-α. Hematopoieticcells plated along in serum-supplement medium without IL-3 or EGF lostviability within forty-eight to seventy-two hours. Other control flaskscontaining monolayers of stromal cells alone had fewer than 0.1% of thecells in the culture supernatant.

Adhesion to Stroma Is Mediated By EGF Receptor Binding To MembraneproTGF-α.

[0044] The specificity of “homing” and adherence of 32D-EGFR cells toGP-TGF-α monolayers was evaluated by testing the ability of EGF andTGF-α to inhibit 32D-EGFR cell binding to the monolayers. Addition of0.04-1.7 nM EGF from the first day of cocultivation prevented theformation of 32D-EGFR cell islands in a dose-dependent manner for atleast seven days (FIG. 7A). The non-adherent 32D-EGFR cells in theculture medium proliferated in the presence of added EGF (FIG. 7B). Thenumber of viable cells harvested per flask increased with increasingconcentration of added EGF. The effect of exogenous TGF-α on coculturesof 32D-EGFR cells and GP-TGF-α cells was similar to the effect of EGF.There were no detectable adherent cell islands by day nine in coculturessupplemented with-12 nM TGF-α.

[0045] The ability of exogenous EGF to induce the disappearance ofpreformed 32D-EGFR cell islands was also evaluated. EGF was added toseven day old cocultures of 32D-EGFR and GP-TGF-α cells that containedmultiple islands. EGF had markedly decreased the number of islandswithin forty-eight hours after addition (FIG. 7C).

[0046] In other experiments, anti-EGF receptor serum or preimmune serumwere added daily to 32D-EGFR/GP-TFG-α cocultures from the first day ofcocultivation. Cocultures containing preimmune serum generated islandsand sustained proliferation of 32D-EGFR cells whereas no adherentislands were detectable and only 3% of the hematopoietic cells wereviable in seven day old cocultures containing anti-EGF receptorantibodies (Table 1). TABLE 1 Effect of anti-EGF receptor antiserum onthe adhesion of 32D-EGFR. cells Serum added Islands/dish Viablecells/dish Preimmune 69 1.42 × 10⁵ Anti-EGFR  0 0.05 × 10⁵

[0047] Confluent monolayers of GP-TGF-α cells in 35 mm dishes werecocultured with 2×10⁶ 32D-EGFR cells per dish. Rabbit preimmune serum oranti-EGF receptor serum was added daily to these cocultures at 1:1000dilution. The number of adherent cell islands/dish and viablenon-adherent cells/dish was scored on day 7 after initiation of thecocultures. Results are the mean of triplicate dishes.

Proliferation of Hematopoietic Cells In Contact With Stroma

[0048] The sustained increase in 32D-EGFR cell number observed incocultures with GP-TGF-α cells could be due to proliferation ofnon-adherent cells released from the adherent islands. Although thelevel of soluble TGF-α in the conditioned medium of GP-TGF-α cells wasbelow the 20 pM detection limit of our assays, generation of a smallamount of TGF-α by cleavage of membrane proTGF-α might be sufficient tostimulate non-adherent cells located near the GP-TGF-α cell monolayer.Alternatively, mitogenic stimulation of 32D-EGFR cells could occur whilethey were anchored to the monolayers via membrane proTGF-α.

[0049] To distinguish between these two possibilities, 5-bromo-2′deoxyuridine (BUdR) was added to nine day old and twenty-one day oldcocultures and allowed to incorporate into relicating DNA for variouslengths of time. To visualize and quantiate cells that had undergone DNAreplication during the time of exposure to BUdR, monolayers containingadherent 32D-EGFR cells and supernatants containing non-adherent cellswere fixed and stained for indirect immunofluorescence with anti-BUdRandibody and rhodamine-conjugated secondary antibody. The stromal cellswere essentially quiescent with fewer than 0.5% of the nuclei becominglabeled under any of the conditions tested (FIG. 8 and Table 2). Asignificant proporation of adherent 32D-EGFR cells incorporated BUdRinto the nucleus (FIG. 8 and Table 2). In nine day old cocultures, thisproportion increased progressively with time of exposure to BUdR,reaching 38% of the adherent cells after a twenty-four hour exposure toBUdR. In twenty-one day old cocultures, as many as 30% of the cellsbecame labeled after only three hours of exposure to BUdR, but thisproportion increased slowly with extended labeling times (Table 2). Incontrast to the high labeling index observed in adherent cells, only 10%or fewer of the non-adherent cells recovered fromn the cocultures becamelabeled (Table 2). This number included any cells that detached from themonolayers during collection of the media at the end of the labelingperiod. Furthermore, removal of the non-adherent cells from the culturesbefore a short (three hours) labeling of the cell layers with BUdR hadessentially no effect on the proportion of adherent 32D-EGFR cellscontaining labeled nuclei (Table 2). From these results, it wasconcluded that 32D-EGFR cells replicated their DNA while they were boundto membrane proTGF-α on the stromal cell monolayer. TABLE 2 DNAreplication in adherent and non-adherent 32D-EGFR cells % of labelednuclei^(a) Coculture age Exposure to BUdR Adherent Non-adherent (days)(hours) cells cells  9  3  8 NT^(c)  9 21 7 12 25 8 24 38 6 21  3 30 11   3^(b) 29 — 12 37 NT 24 48 NT

EXAMPLE 2 RECOVERY OF DONOR ORIGIN STROMAL (GPTGFα) AND HEMATOPOIETIC(32DEGFR) CELLS FROM TRANSPLANTED MICE

[0050] The presence of donor origin stromal cells were detected byplating fresh bone marrow from control and transplanted mice (9 weeksafter transplant with GPTGFα cells and 1 week after a second transplantof 32D-EGF-R cells) at 5×10⁵ cells/60 mm dish and selecting with 300μg/ml of G418. Results are shown in Table 3 as the mean=SD of 3plates/hind limb from each of the three to five mice per group.

[0051] The presence and homing of the donor origin 32D-EGF-R cells wasdetected by plating fresh bone marrow cells from control andtransplanted mice at 5×10⁶ cells/60 mm dish and selecting with 25 μg/mlmycophenolic acid and 30 ng/ml EGF-R. Binding of ¹²⁵I-labelled EGF wasassayed. Equivalent numbers of cells were washed and incubated with¹²⁵I-labelled EGF. The extent of nonspecific binding was measured byincubating cells in the presence of a 100-fold excess of unlabelled EGFand these values were subtracted from bound counts. The results are setforth in Table 3. The data are mean=SD of triplicate determinations/hindlimb from each of three to five mice/group. Other controls were FBM fromC57BL/6 mice (1.5=0.86 cpm/10⁵ cells) 32D cl 3 cells (<0.1 cpm/10⁵cells) and 32D-EGF-R cells (171.4=12.5 cpm/10 cells).

[0052] The data indicates that C57BL/6J mice are stably engrafted invivo with a clonal stromal cell line producing recombinant TGFα(GP-TGFα) to provide an in vivo microenvironment to which 32D-EGFR cellswill home. As shown in Table 3, mice prepared for high dose irradiationof both hind limbs and a total body irradiation dose which was sublethalwere engrafted in vivo by intravenous injection of the GP-TGFα cellline. Six months later, the animals received a total body irradiationdose and injection intravenously, of 32D-EGFR cells. Donor originstromal cells were then measured several week later by explant ofadherent cells showing neo® resistance (a selection marker linked to theTGFα construct) and hematopoietic cells showing mycophenolic acidresistance (a resistance gene linked to the EGFR construct). Thecontrols included animals irradiated and injected with the stromal cellline only, and other animals irradiated and injected with the TGFαproducing stromal cell line and then with the 32D cell line that did nothave the EGFR receptor. The results showed that only in combination ofGP-TGFα cell line engrafted, and then 32D EGFR inoculation, was thereevidence for survival of 32D-EGFR cells in vivo. Furthermore, thesecells were only detected at sites of stable engraftment of GP-TGFαstromal cells. Mice not injected with the stromal cell line, butinoculated with 32-EGFR cells, showed no detectable hematopoietic cellsat the same time interval. Thus, the data provide in vivo evidence forthe engraftment of stromal cell lines followed by homing ofhematopoietic stem cell lines using the receptor ligand interaction asthe mechanism for homing of hematopoietic to the stromal cells in TABLE3 TOTAL NUMBER (% RESISTANT)* HEMATOPOIETIC CELLS 125_(I) - EGF STROMALCELLS Cells/limb bound G418^(I) Mycophenolic acid^(I) × cpm/10⁵ MICECFU-F/LIMB 10⁵ cells Control RHL 2.0 ± 1.9 0.8 ± 0.45 0.7 ± 0.1 −GPTGFα(3.0 ± 2.9%) (2.2 ± 1.2%) +32DEGFR LHL 0 1.3 ± 0.96 N.T. (0) (3.6 ±2.6%) TRANS- RHL 11.3 ± 1.7** 9.2 ± 3.4 4.7 ± 0.16 PLANTED +GPTGFα (17.3± 2.6% (30 ± 11%) +32DEGFR LHL 11.0 ± 3.0** 15.2 ± 8.0 N.T. (16.9 ±4.6%) (51 ± 26%)

Equivalents

[0053] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described specifically herein.Such equivalents are intended to be encompassed in the scope of thefollowing claims.

1. A method for homing hematopoietic stem cells to bone marrow stromalcells in a host, comprising: administering to the hostgenetically-engineered hematopoietic stem cells capable of expressing afirst member of a ligand-receptor binding pair under conditions wherebybinding of the first member of the ligand-receptor binding pair to asecond member of the ligand-receptor binding pair, present on stromalcells, occurs thereby homing the stem cells to the stromal cells.
 2. Amethod for homing hematopoietic stem cells to bone marrow stromal cellsin a host, comprising: administering to the host stromal cells capableof expressing a first member of a ligand-receptor binding pair; andsubsequently administering to the host hematopoietic stem cells capableof expressing a second member of a ligand-receptor binding pair underconditions whereby binding of the ligand to the receptor occurs therebyhoming the stem cells to the stromal cells, wherein either the stromalcells or the hematopoietic stem cells are genetically-engineered toprovide the capability of expressing the appropriate ligand or receptor.3. A method according to claim 2 wherein the ligand-receptor bindingpair is selected from the group consisting transforming growth factorand transforming growth factor receptor or epidermal growth factorreceptor; epidermal growth factor and epidermal growth factor receptor;tumor necrosis factor-α and tumor necrosis factor-receptor; interferonand interferon receptor; platelet derived growth factor and plateletderived growth factor receptor; transferrin and transferrin receptor;avidin and biotin or antibiotin; antibody and antigen pairs; interleukinand interleukin receptor; granulocyte-macrophage colony stimulatingfactor and granulocyte-macrophage colony receptor; macrophage colonystimulating factor and macrophage colony stimulating factor receptor;granulocyte colony stimulating factor and granulocyte colony stimulatingfactor receptor; and sugar molecules and sugar receptors.
 4. A methodaccording to claim 2 wherein the hematopoietic stem cells are 32D or anon-adherent stem cell derived from a human long term bone marrowculture.
 5. A method according to claim 2 wherein the bone marrowstromal cells are selected from the group consisting of GBL/6, KM101,KM102, KM103, KM104 and K105.
 6. A method according to claim 2 whereinthe stromal cells are capable of expressing a ligand.
 7. A methodaccording to claim 6 wherein the ligand is pro-transforming growthfactor-α.
 8. A method according to claim 2 wherein the hematopoieticstem cells are capable of expressing a receptor.
 9. A method accordingto claim 8 wherein the receptor is an epidermal growth factor receptor.10. A method according to claim 2 wherein either thegenetically-engineered stromal cells or hematopoietic stem cells areproduced by transfecting the cells with a retroviral vector containingRNA which is reverse transcribed to DNA encoding a member of aligand-receptor binding pair.
 11. A method for transplanting bone marrowin a host, comprising: administering to the host stromal cells capableof expressing a first member of a ligand-receptor binding pair; andadministering to the host hematopoietic stem cells capable of expressinga second member of a ligand-receptor binding pair under conditionswhereby binding of the ligand to the receptor occurs thereby homing thestem cells to the stromal cells, wherein either the stromal cells orhematopoietic stem cells are genetically-engineered to provide thecapability of expressing the appropriate ligand or receptor.
 12. Amethod according to claim 11 wherein the ligand is pro-transforminggrowth factor-α and the receptor is an epidermal growth factor receptor.13. A method according to claim 11 wherein the hematopoietic stem cellsare selected from the group consisting of 32D or a non-adherent stemcell derived from a human long term bone marrow culture.
 14. A methodaccording to claim 11 wherein the bone marrow stromal cells are selectedfrom the group consisting of GBL/6, KM101, KM102, KM103, KM104 andKM105.
 15. A method according to claim 11 wherein the stromal cells arecapable of expressing a ligand.
 16. A method according to claim 15wherein the ligand is transforming growth factor-α.
 17. A methodaccording to claim 11 wherein the genetically-engineered hematopoieticstem cells are capable of expressing a receptor.
 18. A method accordingto claim 17 wherein the receptor is an epidermal growth factor receptor.19. A method according to claim 11 wherein the genetically-engineeredstromal cells or the hematopoietic stem cells are produced bytransfecting the cells with a retroviral vector containing RNA which isreverse transcribed to DNA encoding a member of a ligand-receptorbinding pair.
 20. A method of treating a host afflicted with a diseaseassociated with a disorder of the bone marrow, comprising: administeringto the host a therapeutically effective amount of stromal cells capableof expressing a first member of a ligand-receptor binding pair; andadministering to the host a therapeutically effective amount ofhematopoietic stem cells capable of expressing a second member of aligand-receptor binding pair under conditions whereby binding of theligand to the receptor occurs, thereby homing the stem cells to thestromal cells, wherein either the stromal cells or hematopoietic stemcells are genetically-engineered to provide the capability of expressingthe appropriate ligand or receptor.